Purification, characterization, and structural elucidation of the active moiety of the previously called "suppressor activating factor (SAF)".
Upon extensive purification of the serum-free supernatant produced by a mutant T cell line (6T-CEM), an immunosuppressive activity was found to reside in an oxidized product of spermine, spermine dialdehyde (SDA). The activity was purified to homogeneity from a serum-free supernatant by using gel filtration chromatography and reverse-phase C18 HPLC. Fast Atom Bombardment (FAB) mass spectral analysis revealed its MW to be 202 and Electron Impact (EI) analysis of the acetylated material identified the purified molecule to be spermine. In the presence of human or rodent plasma, spermine exhibited no immunosuppressive activity up to 2 mg/ml. However, when assayed in the presence of FCS, which contains polyamine oxidase (PAO), spermine is oxidized to its corresponding dialdehyde which is active at 0.1 microM/ml. We have previously described a high molecular weight suppressor activating factor (SAF) found in the serum-containing supernatant of the 6T-CEM cell line. Our preliminary biological data suggest that SDA is probably responsible for the immunosuppressive activities previously observed for the SAF. The strong affinity of SDA for proteins and thiocompounds may account for the apparent high MW previously reported for SAF.
Lau
C
C
Biological Research Department, R. W. Johnson Pharmaceutical Research Institute, Ortho Pharmaceutical Ltd., Don Mills, Ontario, Canada.
Stanojev
D
D
Visconti
V
V
Pang
H
H
Krepinsky
G
G
Grey
A
A
Wang
E
E
Ishaque
A
A
D000447:Aldehydes; D000818:Animals; D002460:Cell Line; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D003470:Culture Media; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D051379:Mice; D008970:Molecular Weight; D013096:Spermine / Q000031:analogs & derivatives*; D013491:Suppressor Factors, Immunologic / Q000302:isolation & purification*; D013601:T-Lymphocytes / Q000032:analysis*
D000447:Aldehydes; D003470:Culture Media; D013491:Suppressor Factors, Immunologic; D013096:Spermine